Killing of NIT-1 islet cells by islet antigen specific 8.3 CD8+T cells in the absence or presence of exogenous IGRP peptide. (A) The killing capacity of in vitro-activated IGRP-specific CD8+ 8.3 T cells was tested. NIT-1 cells or NIT-1 cells plus IGRP peptide at 20ug/ml (final concentration) were used as target cells. NIT-1 cells at the density of 2x103 cells/well (50ul), equal to 4x104 cells/ml, were prepared initially. Effector/Target cell (CD8 8.3 T cells/NIT-1) ratios of 10/1, 5/1, 2/1, and 1/1 were used in a final volume of 200 μl per well. After overnight incubation, 8.3 T cells were removed by washing three times with cell culture medium while adherent NIT-1 cells remained in the testing wells. Subsequently, the ATP contents reflecting the NIT-1 cell remaining viable were measured by the addition of 200 μl of 50% Cell Titer Glo followed by measurement of luminescent signals on a GloMax multi-detection system (Promega). Cytotoxicity (% killing of NIT-1 cells) was calculated as described in the material and method. Shown are mean and SD of 10 replicates for each sample. *p < 0.05; **p < 0.01 (t-test). The data are the representative of three experiments. (B) Based on Figure 1, the numbers of NIT-1 cells were selected for testing in the cytotoxicity assay. NIT-1 cells at 2x103/well, 5x103/well, 1x104/well, and 2x104/well were tested with the E/T ratio of 5/1. After overnight incubation, 8.3 CD8+ T cells were removed by washing three times with cell culture medium while adherent NIT-1 cells remained in the testing wells. Subsequently, cytotoxicity (% killing of NIT-1 cells) was measured and calculated as described in the materials and methods. Shown are the mean and SD of 10 replicates for each sample. The results are the representative of three experiments.